Design and fabrication of smart supramolecular peptide systems is an effective strategy to develop antibacterial agents that can be selectively activated/inactivated by external stimuli for combating bacterial resistance. In this work, we selectively synthesized isomeric tripeptides (i.e., Ala-Gly-Gly-OH and Gly-Gly-Ala-OH) with the simplest structures to construct a minimalistic dual-responsive supramolecular antibacterial system. To impart stimuli-responsiveness, the tripeptides were modified using a hydrophobic n-butylazobenzene tail at the N-terminal, which benefited the enhancement of the hydrophobicity of the tripeptides and they served as synergistic antibacterial moieties. Two different self-assembled 1-D morphologies (i.e., nanotwists and nanofibers) were observed under the same conditions when the position of the Ala residue was altered. More importantly, the supramolecular tripeptide amphiphiles exhibited a reversible assembly/disassembly process in response to different stimuli (i.e., light and host-guest chemistry). Based on the stimuli-responsiveness, the antibacterial/antibiofilm activities against either Gram-negative or Gram-positive bacteria could be reversibly modulated.